Self renewal is one of the two defining properties of a stem cell, and understanding the mechanisms that govern self renewal is among the most important problems in stem cell biology. Unlike mouse, very little is known about the self renewal program of human embryonic stem cells (hESCs), which require undefined or extremely complex culture conditions. We have recently found a simple way to culture mouse, rhesus and human ES cells using a low concentration of butyrate which, in hESCs, activates an alternative and molecularly distinct transcriptional program. We hypothesize that the distinct transcriptional profile induced by butyrate is causally linked to, rather than reflective of, the butyrate self renewal program. This Project proposes to use butyrate and other recent developments from our lab to probe the molecular underpinnings of hESC self renewal. In Specific Aim 1 we will distinguish whether butyrate selects for self renewing hESCs or reprograms hESCs In Specific Aim 2 we will examine whether butyrate's effects on hESCs are reversible. In Specific Aim 3 we will identify butyrate responsive genes that are necessary or sufficient for hESC self renewal. In Specific Aim 4 we will examine specific genomic loci that appear to be coordinately regulated by butyrate.